Computer-assisted Sperm Analysis Research Articles

Analysis of selected sperm samples by a computer-assisted system with high frame rate: A prospective study.

Due to the rapid motility of the selected sperm, sperm parameters cannot be accurately determined by the manual method. So, the application of a computer-assisted sperm analysis system with a high frame rate (HFR-CASA, 85 Hz) in sperm selection is investigated. A total of 177 semen samples were collected for sperm selection with density gradient centrifugation. Then, the manual method and HFR-CASA method will be used to observe and analyze the sperm concentration, motility, and percentage of progressively motile sperm (PR) of the selected sperm samples. The quality control of sperm concentration was performed with microballoons. Two selected sperm samples were analyzed 10 times repeatedly to evaluate the accuracy of the HFR-CASA. The results of microballoons analyzed with the HFR-CASA were in control. The coefficients of variation of sperm concentration, motility, and PR from two selected sperm samples were all below 10%. The results of 177 selected sperm samples analyzed by the manual method and HFR-CASA showed that although there were significant positive correlations in sperm concentration, motility, and PR between them (p < 0.001), the manual method significantly underestimated sperm concentration (p = 0.002) but overestimated sperm motility and PR (p < 0.001). When sperm concentration was below 50 × 106/mL, the manual method significantly overestimated sperm concentration (p < 0.05). However, when sperm concentration was more than 100 × 106/mL, the manual method significantly underestimated sperm concentration (p < 0.001). Regardless of sperm concentration, the manual method consistently overestimated sperm motility and PR (p < 0.001). When sperm concentration was more than 20 × 106/mL, there was no correlation in sperm PR between them (p > 0.05). When sperm concentration was below 50 × 106/mL, the correct rate of captured sperm by the HFR-CASA was more than 98%. The HFR-CASA method is more accurate than the manual method in analyzing the selected sperm samples.

In vitro fertilization with frozen-thawed blue catfish (Ictalurus furcatus) sperm and implications for gene banking

Catfish farming constitutes about 75% of total U.S. finfish aquaculture production, for which the channel catfish female (Ictalurus punctatus) × blue catfish male (I. furcatus) hybrid accounts for nearly 60% of the harvest. Current hatchery technology to produce hybrids is labor intensive and requires the sacrifice of males. This is expensive and time-consuming, as blue catfish males require 4 to 7 years to mature. Sperm, even from mature fish, are often of low quality and quantity and do not necessarily give high fertility and hatch. Therefore, paternal gametes should be used sparingly, using the minimum quantity of sperm for fertilization, while maximizing offspring production. This is particularly important when using cryopreserved cells, as each males' sperm is in limited supply. To optimize the use of available sperm cells, the objectives of this study were to (i) compare sperm swimming kinematics and sperm health metrics before and after cryopreservation; (ii) determine the minimum quantity of frozen-thawed sperm required to maximize hatching success; and (iii) decipher how early offspring development is affected when eggs are sired with fresh and frozen-thawed sperm. Sperm kinematics were evaluated by computer-assisted sperm analysis, while health metrics (i.e., viability, oxidative stress, DNA fragmentation) were assessed using fluorescent imaging and flow cytometry. Over 2 spawning seasons, eggs from 18 females were collected and fertilized using cryopreserved sperm from 36 males at 10 sperm-to-egg ratios (ranging from 1.0 × 104 to 9.0 × 105 sperm per egg). Embryos sired with fresh and frozen-thawed sperm were then incubated under common environmental conditions and growth performance was documented over 40 days post-hatch. Generally, sperm kinematic traits [curvilinear velocity (VCL), motility, progressive VCL, and progressive motility] and health metrics (cell membrane viability, oxidative stress, and DNA fragmentation) declined after cryopreservation. Despite this decrease in sperm quality, hatching success using cryopreserved sperm was high, but dependent on the sperm-to-egg ratio. For instance, at ratios of 1.0 × 104 to 5.0 × 104 sperm per egg, hatch increased from 18.0% ± 8.5 to 43.9% ± 8.2. Adding >5.0 × 104 sperm per egg had no significant effect on hatching success. Furthermore, there were generally no differences in morphology or deformity rates in offspring sired with cryopreserved sperm, compared to a fresh control. These data improve understanding of frozen-thawed sperm quality for blue catfish to improve reproductive sustainability and reduce production costs.

Biallelic variants in IQCN cause sperm flagellar assembly defects and male infertility.

What is the effect of defects in the manchette protein IQ motif-containing N (IQCN) on sperm flagellar assembly? Deficiency in IQCN causes sperm flagellar assembly defects and male infertility. The manchette is a transient structure that is involved in the shaping of the human spermatid nucleus and protein transport within flagella. Our group recently reported that the manchette protein IQCN is essential for fertilization. Variants in IQCN lead to total fertilization failure and defective acrosome structure phenotypes. However, the function of IQCN in sperm flagellar assembly is still unknown. Fifty men with infertility were recruited from a university-affiliated center from January 2014 to October 2022. Genomic DNA was extracted from the peripheral blood samples of all 50 individuals for whole-exome sequencing. The ultrastructure of the spermatozoa was assessed by transmission electron microscopy. Computer-assisted sperm analysis (CASA) was used to test the parameters of curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP). An Iqcn knockout (Iqcn-/-) mouse model was generated by CRISPR-Cas9 technology to evaluate sperm motility and the ultrastructure of the flagellum. Hyperactivation and sperm fertilizing ability were assessed in a mouse model. Immunoprecipitation followed by liquid chromatography-mass spectrometry was used to detect IQCN-binding proteins. Immunofluorescence was used to validate the localization of IQCN-binding proteins. Biallelic variants in IQCN (c.3913A>T and c.3040A>G; c.2453_2454del) were identified in our cohort of infertile men. The sperm from the affected individuals showed an irregular '9 + 2' structure of the flagellum, which resulted in abnormal CASA parameters. Similar phenotypes were observed in Iqcn-/- male mice. VSL, VCL, and VAP in the sperm of Iqcn-/- male mice were significantly lower than those in Iqcn+/+ male mice. Partial peripheral doublet microtubules (DMTs) and outer dense fibers (ODFs) were absent, or a chaotic arrangement of DMTs was observed in the principal piece and end piece of the sperm flagellum. Hyperactivation and IVF ability were impaired in Iqcn-/- male mice. In addition, we investigated the causes of motility defects and identified IQCN-binding proteins including CDC42 and the intraflagellar transport protein families that regulate flagellar assembly during spermiogenesis. More cases are needed to demonstrate the relation between IQCN variants and phenotypes. Our findings expand the genetic and phenotypic spectrum of IQCN variants in causing male infertility, providing a genetic marker for sperm motility deficiency and male infertility. This work was supported by the National Natural Science Foundation of China (81974230 and 82202053), the Changsha Municipal Natural Science Foundation (kq2202072), the Hunan Provincial Natural Science Foundation (2022JJ40658), and the Scientific Research Foundation of Reproductive and Genetic Hospital of CITIC-Xiangya (YNXM-202114 and YNXM-202201). No conflicts of interest were declared. N/A.

Pharmacological Evidence Suggests That Slo3 Channel Is the Principal K+ Channel in Boar Spermatozoa.

Sperm ion channels are associated with the quality and type of flagellar movement, and their differential regulation is crucial for sperm function during specific phases. The principal potassium ion channel is responsible for the majority of K+ ion flux, resulting in membrane hyperpolarization, and is essential for sperm capacitation-related signaling pathways. The molecular identity of the principal K+ channel varies greatly between different species, and there is a lack of information about boar K+ channels. We aimed to determine the channel identity of boar sperm contributing to the primary K+ current using pharmacological dissection. A series of Slo1 and Slo3 channel modulators were used for treatment. Sperm motility and related kinematic parameters were monitored using a computer-assisted sperm analysis system under non-capacitated conditions. Time-lapse flow cytometry with fluorochromes was used to measure changes in different intracellular ionic concentrations, and conventional flow cytometry was used to determine the acrosome reaction. Membrane depolarization, reduction in acrosome reaction, and motility parameters were observed upon the inhibition of the Slo3 channel, suggesting that the Slo3 gene encodes the main K+ channel in boar spermatozoa. The Slo3 channel was localized on the sperm flagellum, and the inhibition of Slo3 did not reduce sperm viability. These results may aid potential animal-model-based extrapolations and help to ameliorate motility and related parameters, leading to improved assisted reproductive methods in industrial livestock production.

Defining an Optimal Range of Centrifugation Parameters for Canine Semen Processing.

Our objective was to determine a clinically relevant range of centrifugation parameters for processing canine semen. We hypothesized that higher gravitational (g) force and longer time of centrifugation would result in improved spermatozoa recovery rate (RR) but poorer semen quality. Cooled storage under standard shipping conditions was used as a stressor to evaluate long-term treatment effects. Individual ejaculates collected from 14 healthy dogs were split into six treatment groups (400 g, 720 g, and 900 g for 5 or 10 min). Sperm RR (%) was calculated post-centrifugation, and plasma membrane integrity (%, Nucleocounter® SP-100™), total and progressive motility (%, subjective and computer-assisted sperm analysis), and morphology (%, eosin-nigrosin staining) were assessed on initial raw semen (T0), post-centrifugation (T1), and 24 h (T2) and 48 h (T3) after cooling. Sperm losses were minimal, and RRs were similar across treatment groups (median >98%, p ≥ 0.062). Spermatozoa membrane integrity was not different between centrifugation groups at any time point (p ≥ 0.38) but declined significantly during cooling (T1 vs. T2/T3, p ≤ 0.001). Similarly, total and progressive motility did not differ across treatments but declined in all groups from T1 to T3 (p ≤ 0.02). In conclusion, our study showed that centrifugation within a range of 400 g-900 g for 5-10 min is appropriate for processing canine semen.

Sperm morphological abnormalities in autosomal dominant polycystic kidney disease are associated with the Hippo signaling pathway via PC1.

Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary kidney disorder mostly caused by mutations in PKD1 or PKD2 genes. Here, we report thirteen ADPKD males with infertility and investigated the sperm morphological defects associated with PC1 disruption. Targeted next-generation sequencing was performed to detect PKD1 variants in patients. Sperm morphology was observed by immunostaining and transmission electron microscopy, and the sperm motility was assessed using the computer-assisted sperm analysis system. The Hippo signaling pathway was analyzed with by quantitative reverse transcription polymerase chain reaction (qPCR) and western blotting in vitro. The ADPKD patients were infertile and their sperm tails showed morphological abnormalities, including coiled flagella, absent central microtubules, and irregular peripheral doublets. In addition, the length of sperm flagella was shorter in patients than in controls of in in. In vitro, ciliogenesis was impaired in Pkd1-depleted mouse kidney tubule cells. The absence of PC1 resulted in a reduction of MST1 and LATS1, leading to nuclear accumulation of YAP/TAZ and consequently increased transcription of Aurka. which might promote HDAC6-mediated ciliary disassembly. Our results suggest the dysregulated Hippo signaling significantly contributes to ciliary abnormalities in and may be associated with flagellar defects in spermatozoa from ADPKD patients.

Mutation of S461, in the GOLGA3 phosphorylation site, does not affect mouse spermatogenesis.

Golgin subfamily A member 3 (Golga3), a member of the golgin subfamily A, is highly expressed in mouse testis. The GOLGA3 protein, which contains eight phosphorylation sites, is involved in protein transport, cell apoptosis, Golgi localization, and spermatogenesis. Although it has been previously reported that nonsense mutations in Golga3 cause multiple defects in spermatogenesis, the role of Golga3 in the testis is yet to be clarified. Immunofluorescence co-localization in cells and protein dephosphorylation experiments were performed. Golga3 S461L/S461Lmice were generated using cytosine base editors. Fertility tests as well as computer-assisted sperm analysis (CASA) were then performed to investigate sperm motility within caudal epididymis. Histological and immunofluorescence staining were used to analyze testis and epididymis phenotypes and TUNEL assays were used to measure germ cell apoptosis in spermatogenic tubules. Immunofluorescence co-localization showed reduced Golgi localization of GOLGA3S465L with some protein scattered in the cytoplasm of HeLa cells .In addition, protein dephosphorylation experiments indicated a reduced band shift of the dephosphorylated GOLGA3S465L, confirming S461 as the phosphorylation site. Golga3 is an evolutionarily conserved gene and Golga3 S461L/S461Lmice were successfully generated using cytosine base editors. These mice had normal fertility and spermatozoa, and did not differ significantly from wild-type mice in terms of spermatogenesis and apoptotic cells in tubules. Golga3 was found to be highly conserved in the testis, and GOLGA3 was shown to be involved in spermatogenesis, especially in apoptosis and Golgi complex-mediated effects. Infertility was also observed in Golga3 KO male mice. Although GOLGA3S465Lshowed reduced localization in the Golgi with some expression in the cytoplasm, this abnormal localization did not adversely affect fertility or spermatogenesis in male C57BL/6 mice. Therefore, mutation of the S461 GOLGA3 phosphorylation site did not affect mouse spermatogenesis.

Effect of various pH levels on the sperm kinematic parameters of boars

The objective of this work was to identify the effects of pH on the structure of kinematic sperm subpopulations using commercial doses of boar semen diluted in Androstar Plus extender. We adjusted the pH value of the diluted semen samples to 7.4, 7.6, 7.8, and 8.0, and evaluated motility parameters using an open-source computer-assisted sperm analysis system. We evaluated the dataset of the kinematic parameters of the individual sperm for the effects of pH on the average values overall and on the subpopulations of sperm. To identify these subpopulations, we reduced the dimensionality of the dataset through principal component analysis followed by hierarchical clustering. We then compared the results for the kinematic parameters across the dataset and in the subpopulations with the average of the corresponding control (i.e., the basal pH value). We found that increasing the pH did not affect the proportion of motile sperm but did affect the manner in which they moved, rendering their movement more linear. The progressive motility increased relative to the control at all pH values above 7.1, while the proportion of sperm with low motility increased at pH 7.8. We distinguished three sperm subpopulations. The proportion of sperm in Subpopulation 1 increased at pH values of 7.8 and 8.0, with the sperm being characterized by low velocity and high values for linearity. The sperm in Subpopulation 3, by contrast, were relatively few but showed relatively high curvilinear velocity. In this study, we demonstrated the presence of subpopulations of boar sperm that responded differentially to changes in pH.

A new computational approach, based on images trajectories, to identify the subjacent heterogeneity of sperm to the effects of ketanserin.

The identification of kinematic subpopulations is of paramount importance to understanding the biological nature of the sperm heterogeneity. Nowadays, the data of motility parameters obtained by a computer-assisted sperm analysis (CASA) system has been used as input to distinct algorithms to identify kinematic subpopulations. In contrast, the images of the trajectories were depicted only as examples of the patterns of motility in each subpopulation. Here, python code was written to reconstruct the images of trajectories, from their coordinates, then the images of trajectories were used as input to a machine learning clustering algorithm of classification, and the subpopulations were described statistically by the motility parameters. Finally, the images of trajectories in each subpopulation were displayed in a way we called Pollock plots. Semen samples of boar sperm were treated with distinct concentrations of ketanserin (an antagonist of the 5-HT2 receptor of serotonin) and untreated samples were used as a control. The motility of sperm in each sample was analyzed at 0 and 30 min of incubation. Six subpopulations were found. The subpopulation 2 presented the highest values of velocities at 0 or 30 min. After 30 min of incubation, the ketanserin increased the values of the curvilinear velocity at high concentrations, whereas the linearity and the straight velocity decreased. Our computational model permits better identification of the kinematic subpopulations than the traditional approach and provides insights onto the heterogeneity of the response to ketanserin; thus, it could significantly impact the research on the relationship between sperm heterogeneity-fertility.

MP01-15 THE IN-VIVO EFFICACY OF LOW-INTENSITY SHOCKWAVE THERAPY ON SPERM PARAMETERS IN AN AGED RAT MODEL

You have accessJournal of UrologyCME1 Apr 2023MP01-15 THE IN-VIVO EFFICACY OF LOW-INTENSITY SHOCKWAVE THERAPY ON SPERM PARAMETERS IN AN AGED RAT MODEL Taher Naeem, Gal Saffati, Weitao Song, Thomas Garcia, Mohit Khera, and Manuel Villarreal Taher NaeemTaher Naeem More articles by this author , Gal SaffatiGal Saffati More articles by this author , Weitao SongWeitao Song More articles by this author , Thomas GarciaThomas Garcia More articles by this author , Mohit KheraMohit Khera More articles by this author , and Manuel VillarrealManuel Villarreal More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003212.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Low-intensity shockwave therapy (LIST) is a safe, non-invasive therapy and an effective treatment option for erectile dysfunction (ED) and many other etiologies by stimulating angiogenesis in the target tissue. It has been theorized that a similar effect could be observed in infertile patients by stimulating spermatogenesis though very little data exists to determine the in-vivo efficacy thereof. This study seeks to investigate the efficacy of LIST on semen parameters in a rat model with age-induced infertility. METHODS: In this primary experiment, 8 Sprague-Dawley (SD) rats at age 6-months were divided into 2 arms: control (N=4) and treatment (N=4). The treatment group received 600 shocks on the testis (3 site on each side, 100 shocks on each site) per session at 0.10 mJ/mm2 3 times a week for 4 weeks and the control group received placebo shocks. One month after the treatment was complete, the rats were euthanized, and semen samples obtained from the epididymis and analyzed using computer assisted sperm analysis. Statistical analysis was done using non-parametric Wilcoxon testing. RESULTS: Testis weights and testosterone levels were not statistically significantly different between the two groups and semen parameters in the treatment group were not statistically significantly improved from the control group. The only notable improvement was in linearity of the average path of the sperm (p=.0239), though this parameter is not a clinically significant one. Histological analysis of the testes and epididymis is ongoing. CONCLUSIONS: This study suggests that LIST does not have an appreciable effect on semen parameters in a SD rat model. This contradicts a prior study that shows an improvement in semen parameters after LIST using a slightly different energy level of 0.05 mJ/mm2. More work needs to be done to determine if LIST is an effective treatment for male infertility. Source of Funding: McNair Foundation © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e8 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Taher Naeem More articles by this author Gal Saffati More articles by this author Weitao Song More articles by this author Thomas Garcia More articles by this author Mohit Khera More articles by this author Manuel Villarreal More articles by this author Expand All Advertisement PDF downloadLoading ...

Effects of Date Palm Pollen Supplementations on The Expression of PRDX1 and PRDX6 Genes in Infertile Men: A Controlled Clinical Trial.

Accumulating evidences suggest that date palm pollen (DPP) induces antioxidant activity and improves semen parameters in male rats. However, there is a few scientific evidences in support of the DPP effects on human male fertility. Hence, the effect of oral consumption of DPP on sperm parameters and expression pattern of Peroxiredoxin- 1 (PRDX1) and Peroxiredoxin-6 (PRDX6) genes was evaluated in men with infertility. The current controlled clinical trial included 40 men with infertility (DPP group) and 10 normospermic fertile men as controls. The DPP group received gelatinous capsules of DPP (400 mg/kg) for 74 days. Semen sampling was done before and after treatment in the both groups. Semen analysis and 8-isoprostane concentration assessments were performed by computer-assisted sperm analysis and ELISA methods, respectively. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays were employed to explore expression of PRDX1 and PRDX6 genes. DPP consumption significantly improved semen volume (P=0.030), count (P<0.001) and morphology of sperm (P=0.023). Concentration of 8-isoprostane was significantly decreased after intervention in the DPP group (P<0.001). DPP consumption led to a significant elevation in the expression of PRDX1 and PRDX6 genes (P<0.001). Elevated gene expression of PRDX6 and PRDX1 was positively correlated with improved parameters of sperm including count, volume, motility and morphology. Taken together, DPP seems to promote sperm quality through a decrease in reactive oxygen species (ROS) by increasing expression of antioxidant genes. Further large-scale studies are required to challenge this hypothesis (registration number: IRCT2015021221014N2).

Sperm physiology and in vitro fertilising ability rely on basal metabolic activity: insights from the pig model

Whether basal metabolic activity in sperm has any influence on their fertilising capacity has not been explored. Using the pig as a model, the present study investigated the relationship of energetic metabolism with sperm quality and function (assessed through computer-assisted sperm analysis and flow cytometry), and fertility (in vitro fertilisation (IVF) outcomes). In semen samples from 16 boars, levels of metabolites related to glycolysis, ketogenesis and Krebs cycle were determined through a targeted metabolomics approach using liquid chromatography-tandem mass spectrometry. High-quality sperm are associated to greater levels of glycolysis-derived metabolites, and oocyte fertilisation and embryo development are conditioned by the sperm metabolic status. Interestingly, glycolysis appears to be the preferred catabolic pathway of the sperm giving rise to greater percentages of embryos at day 6. In conclusion, this study shows that the basal metabolic activity of sperm influences their function, even beyond fertilisation.

Management of dog sperm parameters and gut microbiota composition with Lactobacillus rhamnosus supplementation.

The effects of probiotics supplementation on the reproductive function have been evaluated in many species, but no study has evaluated the changes in the gut microbiome along with the sperm quality changes simultaneously. This study evaluated the effects of dietary supplementation with probiotics on the gut microbiome, sperm quality and gene expression, along with possible correlations between these parameters in dogs. The dogs were supplemented with Lactobacillus rhamnosus for six weeks, and fecal and semen samples were collected at 0, 3, and 6weeks. Fecal samples were assessed using 16S Metagenomic Sequencing for gut microbiome analysis; and semen samples were analyzed using computer-assisted sperm analysis, DNA and acrosome integrity assessment, viability and morphology assessment, and real-time PCR. The analyses suggested that probiotic supplementation improved kinematic parameters, viability, DNA and acrosome integrity, and morphology of sperms. The mRNA levels of genes associated with fertility, DNA repair and integrity, and antioxidation were also upregulated. The sperm parameters were positively correlated with the relative abundance of Actinobacteria, Allobaculum, Phascolarctobacterium and Catenibacterium, and negatively correlated with Faecalibacterium and Streptococcus. Taken together, the sperm quality enhancement through the gut-testis axis may be due to a change in the gut microorganisms populations.

Modulatory effect of MG-132 proteasomal inhibition on boar sperm motility during in vitro capacitation.

A series of biochemical and biophysical changes during sperm capacitation initiates various signaling pathways related to protein phosphorylation leading to sperm hyperactivation, simultaneously with the regulation of proteasomal activity responsible for protein degradation and turnover. Our study aimed to unveil the role of the proteasome in the regulation of boar sperm motility, hyperactivated status, tyrosine phosphorylation, and total protein ubiquitination. The proteolytic activity of the 20S proteasomal core was inhibited by MG-132 in concentrations of 10, 25, 50, and 100 μM; and monitored parameters were analyzed every hour during 3 h of in vitro capacitation (IVC). Sperm motility and kinematic parameters were analyzed by Computer Assisted Sperm Analysis (CASA) during IVC, showing a significant, negative, dose-dependent effect of MG-132 on total and progressive sperm motility (TMOT, PMOT, respectively). Furthermore, proteasomal inhibition by 50 and 100 μM MG-132 had a negative impact on velocity-based kinematic sperm parameters (VSL, VAP, and VCL). Parameters related to the progressivity of sperm movement (LIN, STR) and ALH were the most affected by the highest inhibitor concentration (100 μM). Cluster analysis revealed that the strongest proteasome-inhibiting treatment had a significant effect (p ≤ 0.05) on the hyperactivated sperm subpopulation. The flow cytometric viability results proved that reduced TMOT and PMOT were not caused by disruption of the integrity of the plasma membrane. Neither the protein tyrosine phosphorylation profile changes nor the accumulation of protein ubiquitination was observed during the course of capacitation under proteasome inhibition. In conclusion, inhibition of the proteasome reduced the ability of spermatozoa to undergo hyperactivation; however, there was no significant effect on the level of protein tyrosine phosphorylation and accumulation of ubiquitinated proteins. These effects might be due to the presence of compensatory mechanisms or the alteration of various ubiquitin-proteasome system-regulated pathways.

Effects of preconception exposure to phthalates on mouse sperm capacitation parameters.

Phthalates have been linked to adverse male reproductive health, including poor sperm quality and embryo quality as well as a longer time to pregnancy (months of unprotected intercourse before conception occurs). The present study aimed to evaluate the effect of preconception exposure to two ubiquitous phthalate chemicals, di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DBP), and their mixture on sperm function, fertilization, and embryo development in mice. Adult male C57BL/6Jmice aged 8-9weeks were exposed to di(2-ethylhexyl) phthalate, di-n-butyl phthalate, or their mixture (di-n-butyl phthalate+di(2-ethylhexyl) phthalate) at 2.5mg/kg/day or vehicle for 40days (equivalent to one spermatogenic cycle) via surgically implanted osmotic pumps. Caudal epididymal spermatozoa were extracted and analyzed for motility using computer-assisted sperm analyses. Sperm phosphorylation of protein kinase A substrates and tyrosine phosphorylation, markers of early and late capacitation events, respectively, were analyzed by Western blots. In vitro fertilization was used to evaluate the sperm fertilizing capacity. While the study did not reveal any significant differences in sperm motility and fertilization potential, abnormal sperm morphology was observed in all phthalate exposures, particularly in the phthalate mixture group. In addition, the study revealed significant differences in sperm concentration between control and exposed groups. Moreover, protein phosphorylation of protein kinase A substrates was decreased in the di(2-ethylhexyl) phthalate and mixture exposure groups, while no significant changes in protein tyrosine phosphorylation were observed in any of the groups. Assessment of the reproductive functionality did not reveal significant effects on in vitro fertilization and early embryo development rates but showed wide variability in the phthalate mixture group. Our findings suggest that preconception phthalate exposure affects sperm numbers and phosphorylation of protein kinase A substrates involved in capacitation. Future research is warranted to examine the associations between phthalate exposure and capacitation in human spermatozoa.

The effect of luteolin on spermatological parameters, apoptosis, oxidative stress rate in freezing rabbit semen.

The aim of the present study was to determine the effects of Luteolin (LUT) on semen quality, oxidative stress, apoptosis, acrosomal integrity, mitochondrial membrane potential and dead sperm ratio in rabbits. Ejaculates from six New Zealand rabbits were collected, evaluated and pooled. The pooling was divided into five groups as control (no additive) LUT 25 µM, LUT 50 µM, LUT 100 µM and LUT 200 µM and LUT added. It was then filled into a falcon tube with Tris-based extender at a final concentration of approximately 35 x 106 spermatozoa. Diluated rabbit semen samples were drawn into frozen and thawed. Frozen semen straws were thawed at 37°C in 30 seconds. According to our findings, no statistical difference was found between all doses of luteolin and the control group in the CASA (computer assisted sperm analysis) analysis performed at 4°C. However, total motility, progressive motility and rapid sperm percentage were found to be higher in the frozen and thawed rabbit semen at a dose of LUT 50 µM compared to the other groups (p⟨0.05). While amplitude of lateral head displacement (ALH) and beat cross-frequency (BCF) values were found at the lowest dose of LUT 200 µM, a statistically significant difference was observed between the other groups. When the flow cytometry results were examined, no statistical difference was found between the rate of dead sperm, acrosomal integrity, mitochondrial membrane potential and apoptosis rate. Morever, the H2 O2 percentage was found to be lower in all experimental groups compared to the control group (p⟨0.001). In conclusion, the addition of LUT in long-term storage of rabbit semen provided a protective effect for spermatozoa with its antioxidative properties against damage caused by cryopreservation.

The Quality of Frozen Friesian Holstein Semen after Long-Term Storage

Semen cryopreservation is the long-term storage at very low temperatures in liquid nitrogen for future use. This study investigates the quality of frozen Friesian Holstein (FH) semen after long-term storage. Samples of FH semen stored for 25, 20, 15, 10, 5, and 1 years were collected from one of the national centers for artificial inseminations. Frozen semen was stored in containers with liquid nitrogen at -196 ᵒC in a room with a temperature of 20 ᵒC The variables used after thawing were sperm motility, viability, and abnormalities, as well as plasma membrane integrity (IPM) using computer-assisted sperm analysis (CASA), eosin-nigrosine staining, and hypoosmotic swelling (HOS) test, respectively. Data were analyzed by one-way ANOVA and expressed as mean ± SEM. The result showed no significant differences in sperm viability, abnormalities, and IPM. Furthermore, sperm motility was >40%, consistent with the Indonesian standard for frozen bovine semen. CASA analysis showed that all variables of the motility pattern have no significant difference, except linearity (LIN). The Lin of sperm was lower in frozen semen after one and five years than after 20 and 25 years of storage. The overall quality of semen after 25, 20, 15, 10, 5, and 1 years of storage met the standard and was suitable for artificial insemination.

Acrylamide-induced meiotic arrest of spermatocytes in adolescent mice by triggering excessive DNA strand breaks: Potential therapeutic effects of resveratrol.

Background: Baked carbohydrate-rich foods are the main source of acrylamide (AA) in the general population and are widely consumed by teenagers. Considering the crucial development of the reproductive system during puberty, the health risks posed by AA in adolescent males have raised public concern.Methods: In this study, we exposed 3-week-old male pubertal mice to AA for 4weeks to evaluate its effect on spermatogenesis using computer-assisted sperm analysis (CASA) and historical analysis. Flow cytometric analysis and meiocyte spreading assay were conducted to assess meiosis in mice. The expression of meiosis-related proteins and double-strand break (DSB) proteins were evaluated by immunoblot analyses. Additionally, isolated spermatocytes were used to explore the role of resveratrol in AA-induced damages of meiosis.Results: Our results showed that AA decreased the testicular and epididymal indexes, reduced sperm count and motility, and induced morphological disruption of the testes in pubertal mice. Subsequent meiotic analysis revealed that AA increased the proportion of 4C spermatocytes and decreased the proportion of 1C spermatids. The expression levels of meiosis-related proteins (SYCP3, Cyclin A1 and CDK2) were downregulated, and signaling proteins (γH2AX, p-CHK2 and p-ATM) expression levels were upregulated in AA-treated mice testes. Similar expression patterns were observed in primary spermatocytes treated with AA and these effects were reversed significantly by resveratrol.Conclusions: Our results indicate that AA induces meiotic arrest via persistent activation of DSBs, which may contribute to AA-compromised spermatogenesis. Resveratrol could serve as a potential therapeutic agent against AA-induced meiotic toxicity. These data highlight the importance of natural product supplementation for treating AA-related reproductive toxicity.

Influence of Sperm Number and Antioxidant Melatonin in Extender on the Quality of Post-Thawing Sheep Spermatozoa

This study aimed to examine the effect of spermatozoa concentration and the effectiveness of melatonin supplementation in diluent on the quality of post-thawing semen. Ejaculated semen was collected using the artificial vaginal method (MVB). The study was carried out in two stages, firstly semen was frozen in andromed diluent with different concentrations in one straw (50, 100, 200 million per straw), and the second was frozen semen in diluent supplemented with melatonin with different doses (0, 0,5, 1, 0 and 1.5 mM melatonin). Parameters observed were the movement of spermatozoa using Computer Assisted Sperm Analysis, membrane integrity, and acrosome integrity. Data were analyzed using ANOVA and further tested using Duncan's test. The results showed no significant difference in the quality and movement pattern of sheep semen when frozen at concentrations of 50, 100, or 200 million per straw (P 0.05). Furthermore, adding melatonin to the diluent in this study affected spermatozoa's total motility and progressive motility at a concentration of 1.0 mM (P0.05) but did not significantly affect the percentage of spermatozoa motility pattern characteristics. From the results, it can be concluded that the concentration of per straw spermatozoa does not affect the quality of sheep semen, and melatonin supplementation in diluent can reduce the effects of the frozen-thawed process on motility, acrosome cap, and plasma membrane integrity in sheep semen. Melatonin supplementation with a concentration of 1 mM in the extender was the highest quality concentration in this study.

Hexane fraction of Prunus japonica thunb. Seed extract enhances boar sperm motility via CatSper ion channel

IntroductionMammalian sperm motility is facilitated by flagellar beating, which depends on active ion movement through ion channels and their regulation. Prunus japonica Thunb., also known as oriental bush cherry, is a widely used traditional medicinal plant. However, its significance in improving fertility and sperm quality has not been fully elucidated yet. One of our previous reports revealed that P. japonica seed extract (PJE) can improve human sperm motility through intracellular pH modulation. Aim of the studyThe present study was designed to investigate the effects of PJE on boar spermatozoa and potential underlying mechanisms. Materials and methodsSperm motility changes were examined using a computer-assisted sperm analysis (CASA) system under both capacitated and non-capacitated conditions. Intracellular calcium concentration was measured using either confocal microscopy or a fluorescent microplate reader with Fluo-4AM calcium fluorescent dye. Sperm capacitation-related proteins were analyzed using western blotting. ResultsA significant increase in rapid motility, velocity, and linear displacement of sperm was observed in PJE-treated capacitated boar sperm, whereas the effect was insignificant in the non-capacitated counterparts. Intracellular calcium levels were significantly elevated upon PJE treatment (20–100 μg/L) in a concentration-dependent manner. The increase in intracellular calcium levels was inhibited when the sperm were treated with a CatSper (cation channel of sperm) channel inhibitor, 10 μM Mibefradil, indicating the involvement of the ion channel in the PJE modulatory mechanism. In addition, western blotting revealed an increased level of protein phosphorylation (p-tyrosine and p-PKA), which is a hallmark of sperm capacitation. ConclusionsPJE treatment resulted in a combination of increased motility, intracellular calcium concentration, and capacitation, thereby indicating its potential to ameliorate sperm motility parameters and induce capacitation of boar spermatozoa as a result of intracellular calcium elevation via the CatSper channel. Our observations further elaborate ion channel-related underlying mechanisms and show putative implications of the seed extract of traditionally used P. japonica Thunb. in ameliorating sperm quality.